Francisella tularensis is a facultative intracellular bacterial pathogen that causes serious and potentially life threatening illness by surviving and multiplying within host cells, primarily macrophages. Because the bacterium has extraordinarily high infectivity, causes serious morbidity and mortality, and is easily dispersed, it is also considered a potential agent of bioterrorism. While currently available antibiotics are effective in treating tularemia, F. tularensis can be engineered to carry antibiotic resistance genes. For these reasons, new approaches to treatment and prevention of tularemia are needed. However, devising such strategies requires an improved understanding of the interaction between F. tularensis and its host cells. We have demonstrated that fully virulent F. tularensis enter macrophages via spacious pseudopod loops and that the bacterium then enters a phagosomal compartment that exhibits arrested maturation in that it acquires limited amounts of lysosome associated membrane glycoproteins, does not acquire cathepsin D, and is only minimally acidified prior to escape of the bacterium into the macrophage cytoplasm. We propose to define further the intracellular biology of F. tularensis and to identify the virulence mechanisms that allow it to evade phagosome-lysososome fusion, phagosome acidification, and to escape into the host cell cytoplasm. We shall identify host and bacterial proteins that are important to resistance to phagosome-lysosome fusion and phagosome escape by a mass spectrometry based proteomic comparison of purified phagosomes containing live wild type F. tularensis, killed F. tularensis, and selected F. tularensis mutants that fail to escape and that traffic differently within the host cell. We shall identify bacterial genes required for growth in macrophages that are required for altering the intracellular trafficking and phagosome escape by screening a transposon mutant library. We shall determine whether specific host and bacterial proteins that we identify by these methods are required for uptake of the bacteria by looping phagocytosis, resistance to phagosomelysosome fusion, or phagosome escape by using siRNA techniques and by examining the phenotype of targeted bacterial mutants by immunofluorescence microscopy and transmission electron microscopy. We shall determine whether F. tularensis mutants that are defective in phagosome-lysosome fusion or phagosome escape can be restored to wild-type phenotype by delivering the protein corresponding to the mutated gene into the host cell cytoplasm or into the F. tularensis phagosome. This will provide information regarding the possible site of action of these bacterial proteins.